Cotyledon Explants Research Articles

Enhanced Regeneration Through ex vitro Rooting and Agrobacterium-mediated Genetic Transformation of Eggplant (Solanum melongena L.)

Regeneration of in vitro multiple shoots was achieved through organogenesis on MS supplemented with 2.0 mg/l BAP and 0.5 mg/l Kn from cotyledonary leaf explants of two local varieties of eggplant (Solanum melongena L.). Elongation of regenerated shoots was obtained on growth regulator free MS. In vitro root induction from excised regenerated shoots was less effective on MS with or without plant growth regulators. On the other hand regenerated shoots treated with 10 mM IBA for 5 min were found to be effective for ex vitro rooting in sterilized soil. Following sufficient development of roots, the ex vitro rooted plantlets were acclimatized in growth room condition, and were transferred to the field having 100% survival rate. The regeneration system developed was utilized for Agrobacterium-mediated genetic transformation using Agrobacterium tumefaciens strain LBA4404/pBI121 containing GUS and nptII genes. Adequate transformation response was obtained from cotyledonary leaf segments with bacterial suspension having an optical density of 0.50 at 600 nm with 30 min incubation followed by co-cultivation period of 72 hrs in Nayantara (BARI Begun-5) variety of eggplant. Selection of transformed shoots was carried out on MS supplemented with 2.0 mg/l BAP, 0.5 mg/l Kn, 300 mg/l carbenicillin and 100 mg/l kanamycin. Stable integration of GUS and nptII genes in Nayantara were confirmed through PCR analysis using the genomic DNA isolated from transformed shoots.
 Plant Tissue Cult. & Biotech. 31(1): 97-108, 2021 (June)

A novel and rapid method for Agrobacterium-mediated production of stably transformed Cannabis sativa L. plants

The development of genetically transformed plants is an elusive landmark in Cannabis sativa L. breeding. Despite its economic interest, at present, protocols for producing transgenic C. sativa plants are scarce. We studied the ability of hypocotyl, cotyledon and meristem explants from six C. sativa hemp varieties for transgenic plant regeneration. For this, we firstly evaluated in vitro regeneration rates of hypocotyls cultured in medium without plant growth regulators, and cotyledons cultured in medium supplemented with 0.4 mg L−1 of thidiazuron (TDZ) and 0.2 mg L−1 of α-naphthaleneacetic (NAA). Subsequently, the effect of different kanamycin concentrations (50, 100, 200, 500 and 750 mg L−1) on hypocotyl regeneration rate was determined. Finally, we assessed transformation rates after hypocotyl, cotyledon and meristem co-culture with Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pBIN19 containing the β-glucuronidase (uidA) reporter gene and the kanamycin resistance neomycin phosphotransferase (nptII) genes. Plant transformation was validated through in vitro culture of regenerating shoots in kanamycin-containing selective regeneration medium, by GUS histochemical assay for uidA expression, and by PCR amplification of uidA and nptII genes. Our results showed that hypocotyls reached a higher regeneration rate (53.3 %) than cotyledons (18.1 %) without Agrobacterium co-culture. On the other hand, 100 mg L−1 kanamycin proved to be the best concentration in terms of regeneration rate (63.3 %) and spontaneous rooting rate of hypocotyl regenerating shoots (12.2 %), which displayed a 7.1 % of albinism rate. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyl was the best explant type achieving 23.1 % of regeneration rate, which contrasts with the 1.0 % regeneration rate detected for cotyledons. Transgenic plants were obtained from all explant types evaluated. Although there were significant differences among varieties evaluated, hypocotyls proved to be superior to already-developed meristems, reaching a transformation rate of 5.0 % and 0.8 % respectively. Despite the extremely low regeneration rate of cotyledons after A. tumefaciens co-culture, all cotyledon-derived regenerating shoots analyzed were successfully transformed. Our hormone-free protocol doubles the transformation rate of regenerating shoots, also producing transgenic plants three times faster than other already published protocols. This has relevant implications for C. sativa breeding, enabling not only genetic transformation, but also the use of new plant breeding techniques such as targeted genome editing by using CRISPR/Cas systems. This may foster the development of C. sativa varieties with specific biochemical profiles, or tolerant to biotic and abiotic stresses among others.

Effects of Different β-Lactam Antibiotics on Indirect Tomato (Solanum lycopersicum L.) Shoot Organogenesis and Agrobacterium tumefaciens Growth Inhibition In Vitro.

A β-lactams that act by inhibiting the bacterial cell wall biosynthesis are one of the most common classes of antibiotics applied to suppress the growth of latent bacterial infection associated with the plant tissue culture, as well as in the Agrobacterium-mediated transformation techniques. Plant sensitivity to antibiotics usually is species-, genotype-, or even tissue-specific and mainly depends on concentrations, growth conditions, and culture system. In the presented article, we estimated a comparative effect of four β-lactam antibiotics (Claforan®, timentin, amoxicillin, and Amoxiclav®) at different concentrations in an agar-solidified Murashige and Skoog (MS) culture medium supplemented with 5 mg L−1 6-benzylaminopurine (6-BA) and 0.1 mg L−1 indole-3-acetic acid (IAA) on in vitro callus induction and shoot organogenesis from hypocotyl and cotyledon explants of two tomato cultivars (Rekordsmen, Moryana). The role of clavulanic acid in combination with amoxicillin (Amoxiclav®) in the shoot organogenesis frequency and number of shoots per explant has been demonstrated. Additionally, the growth inhibition of Agrobacterium tumefaciens AGL0 strain according to agar disk-diffusion assay was studied. As a result, both stimulatory (timentin, amoxicillin, and Amoxiclav®) and inhibitory (Claforan®) effects of β-lactam antibiotics on in vitro morphogenetic responses of tomato were noted. It was found that clavulanic acid, which is part of the commercial antibiotic Amoxiclav®, significantly increased the shoot regeneration frequency from cotyledon and hypocotyl explants of Rekordsmen tomato cultivar. Possible reasons for the stimulating effect of clavulanic acid on the induction of shoot organogenesis are discussed. According to agar disk-diffusion assay, the maximum diameter of growth inhibition zones (43.9 mm) was identified using 200 mg L−1 timentin. The in vitro antibacterial activity of tested β-lactam antibiotics was arranged in the following order: timentin > Claforan® > amoxicillin ≥ Amoxiclav®. Thus, to suppress the growth of internal and latent bacterial infection of tomato plant tissue culture, as well as for transformation of Moryana and Rekordsmen cultivars by A. tumefaciens strain AGL0, we recommend adding of 100–200 mg L−1 timentin or 400–800 mg L−1 Amoxiclav® to the shoot induction medium.

Optimization of Different Factors for Initiation of Somatic Embryogenesis in Suspension Cultures in Sandalwood (Santalum album L.)

Santalum album (L.) is a prized tropical tree species of high therapeutic and industrial importance. The wood of these naturally grown plants is extensively harvested to acquire therapeutically important metabolite santalol and be used for additional functions such as in wood statuette industries. Due to high demand, it is crucial to maintain a sufficient plant population. An easy protocol for establishing cell suspension culture initiated from the loose embryogenic callus mass of sandalwood was realized by shifting 6–8-week-old morphogenic calli acquired from the mature embryonic axis and cotyledon explant cultures in fluid media. The asynchronous embryogenic cultures were sloughed with clumps of flourishing cell clumps and embryos of various progressive phases along with diffident non-embryogenic tissues. The frequency of embryo proliferation was evidenced to determinethe expansion pace of embryogenic masses under diverse conditions. The intonation of initiation and creation of cell suspension was under the directive of the influence of exogenous plant growth regulators amended in the nutrient medium at different concentrations and combinations. Maximum relative growth rate (386%) and clumps/embryoids in elevated integers (321.44) were accomplished on MS nutrient medium fortified with 2.0 mg L−1 2,4-D in association with 0.5 mg L−1 BA and 30.0 g L−1 sucrose raised from mature embryonic axis-derived calli. Plantlet regeneration in higher frequency (84.43%) was evidenced on MS medium amended with 1.0 mg L−1 each of TDZ and GA3 in conjunction with 0.5 mg L−1 NAA and 20.0 g L−1 sucrose. Mature embryonic axis-derived calli were found to be constantly better than mature cotyledon-derived calli for raising profitable and reproducible cell suspension cultures. Regenerants displayed normal growth and morphology and were founded successfully in the external environment after hardening.

Development of a Simple and Low-Resource Regeneration System of Two Greek Tomato Varieties

Native tomato landrace varieties (Solanum lycopersicum L.) are sources of high genetic diversity and are adaptable to local environmental conditions. The in vitro propagation system can be used as a tool to produce disease-free, high-quality propagation material and preserve the unique characteristics of commercial Greek tomato varieties, such as the variety Areti and the traditional landrace variety Makedonia, both of exceptional quality and economic importance. Twenty- and 27-day-old cotyledon and leaf explants were cultivated in 4 regeneration media supplemented with 0 or 0.1 mg∙L−1 indole-3-acetic acid and 0.5 or 1 mg∙L−1 zeatin. Regeneration efficiency was dependent on variety, explant type and age, and regeneration media. Areti was a more appropriate target genetic material, exhibiting a six-fold greater response to regeneration compared to Makedonia, regardless of media and explant age. The regenerated shoots of both varieties were successfully rooted (60%) and acclimatized (98%). This regeneration protocol would be valuable in the production of propagation material for commercial and conservation practices and in breeding programs for genetic improvement.

Effect of salinity and drought stress on morphological and biochemical properties of two Iranian fenugreek (Trigonella foenum-graecum) populations

In this study, micro propagation of two Iranian fenugreek populations and their morphological and biochemical responses to salinity and drought stresses in in vitro culture condition were conducted using factorial experiment in a completely randomized design in three replications. Different explant type (terminal bud, cotyledon and epicotyledon explant) were cultured in MS medium contain different concentration of plant growth regulators such as kin (0, 0.5 and 1 mg / l) and 2,4-D (0.5, 1 and 2 mg / l). Murashige and Skoog (MS) medium supplemented with 1 mg/l kinetin and 2 mg/l 2,4-D showed the highest callus proliferation rate per explants in both populations. The highest amount of callus volume was obtained from the explants of the terminal bud. Proliferated calli from terminal bud explant were green and yellowish, from cotyledon were yellowish to white with soft texture, and the cotyledons were greenish and compact. The results of salinity stresses with different concentrations of sodium chloride (0, 70 and 120 mM) and drought stress with polyethylene glycol (0, 5 and 10%) showed that both stresses decreased callus growth and increased total protein, proline, catalase, peroxidase and trigonelin content in both populations. Trigonelin measurement showed that ‘Borazjan’ papulation had higher trigonelin content, in vitro, than ‘Ardestan’ papulation.

An Analysis of the Micropropagation and Peculiarities of Eggplant (Solanum melongena L.) Cultivation Technology

This review is devoted to biotechnological methods and approaches used in eggplant selection. Using in-vitro techniques, micropropagation makes it possible to obtain Solanum melongena L. plants identical to the original ones. This article discusses various aspects of eggplant microcloning; draws conclusions about the successful induction of shoots, which is an important stage required for the regeneration of the callus; and describes the methodological issues of origin, explant treatment, cultivation features, composition of nutrient media, growth regulators, growing conditions and rooting algorithms, and adaptation of the resulting regenerant plants. The microclonal reproduction of Solanum melongena L. by organogenesis from cotyledon and hypocotyl explants is described. After explants were introduced into an in-vitro culture, significant differences in the nature of growth and development of the tissues in the nutrient media were observed on the 6-8th day. A comparative analysis of the stimulating effect of growth regulators revealed that thidiazuron with a concentration of 0.02 mg/l had the highest efficiency; its effect on the regenerative capacity of the explant depended on the concentration in the medium. The dependence of all stages of microclonal reproduction on the genotype is shown.
 Keywords: Solanum melongena L., eggplant, nightshade, microcloning, biotechnology

Adventitious root cultures of Clitoria ternatea L. and its potential as a memory enhancer alternative

Clitoria ternatea L., known as the butterfly pea plant, is from the Fabaceae family and is commonly used in traditional medicine or as a brain tonic in Asia. The root extracts of C. ternatea have been previously studied for its neuroprotective properties linked to memory enhancement of the mammalian brain, indirectly delaying the occurrence of neurodegenerative diseases. The current study aims to establish in vitro adventitious root cultures of C. ternatea via cotyledon explants and to identify the phytoconstituents of the induced adventitious roots. In this study, the highest percentage of induction (95.24%) was achieved in half-strength MS medium supplemented with 2.50 mg/L NAA (1-naphthaleneacetic acid) after 6 weeks of culture. Results also indicated that the supplementation of 1.50 mg/L NAA induced the highest number of adventitious roots (12.86 ± 2.14), while 2.50 mg/L 4-Cl-IAA (4-chloroindole-3-acetic acid) yielded higher adventitious root length (0.75 ± 0.13 cm) in comparison with the other auxin treatments. Microscopic analysis indicated thicker in vitro root morphology for all NAA treatments in comparison with the roots of in vitro seedling. Ethanolic extract of in vitro-generated adventitious roots showed potential anti-acetylcholinesterase activities (IC50 = 1519.89 ± 0.18 μg/mL) albeit being lower than the control (IC50 = 70.37 ± 0.01 μg/mL), while gas chromatography–mass spectrometry analysis identified the presence of pentacyclic triterpenes, phytosterols and fatty acids in the adventitious root culture that are linked to the mammalian neuroprotective properties of C. ternatea. The current investigation reports on the potential of C. ternatea root cultures being further utilized to produce valuable metabolites linked to the mammalian memory enhancement.

Evaluation of bioactive flavonols and ent-kaurenes in the 2,4-dichlorophenoxyacetic acid-induced calli of Senegalia nigrescens using FTIR and GC–MS

The increasing demand for plant-derived pharmacologically active compounds calls for sustainable alternative sources and conservation of medicinal plant species. To this end, the present study investigated the development of a callogenesis protocol for Senegalia nigrescens. The plant growth regulators (PGRs), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ) at concentrations of 0.5 and 1.0 mg L−1 were tested for callus induction in the leaf, stem, cotyledon and root explants of S. nigrescens. 2,4-D was the more effective PGR across the different explant sources with 85 to 100% stem, root and cotyledon explants forming callus (EFC). Hard and compact calli with nodular structures were obtained from the stem explants, the root-derived calli were soft and moist while the cotyledon explants produced glassy and friable calli. Methanol extracts of calli obtained from the stem and root explants were characterised using fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC–MS). The FTIR spectra and GC–MS profiles revealed the presence of the four compounds evaluated, quercetin, quercetin-3-O-methyl ether, ent-kaur-15-en-18-ol and ent-kaur-15-ene-18,20-diol in both 4 and 12-week-old, root-derived calli. This study, therefore, provides preliminary information for the propagation and phytochemical harvesting in S. nigrescens.

Cell Suspension Culture and In Vitro Screening for Drought Tolerance in Soybean Using Poly-Ethylene Glycol.

Soybean (Glycine max (L) Merrill) is used in India mostly as a substantial fund of protein and oil, which makes the crop significantly important. Somaclonal variation has been researched as a base of additional variability for drought in soybean. In the present experiment calli/cell clumps/embryoids rose from immature and mature embryonic axis and cotyledons explants were exposed to different concentrations of polyethylene glycol (PEG6000). A discontinuous method proved to be superior as it permitted the calli/embryoids/cell clumps to regain their regeneration competence. A total of 64 (12.21%) plantlets of genotype JS335 and 78 (13.13%) of genotype JS93-05 were regenerated after four consequent subcultures on the selection medium with an effective lethal concentration of 20% PEG6000, and proliferated calli/embryoids/cell clumps were further subcultured on Murashige and Skoog regeneration medium supplemented with 0.5 mgL−1 each of α-napthalene acetic acid (NAA), 6-benzyladenine (BA) and Kinetin (Kn), 20.0 gL−1 sucrose and 7.5 gL−1 agar. Putative drought-tolerant plantlets were acquired from genotype JS93-05 (38) in more numbers compared to genotype JS335 (26). Random decamer primers confirmed the presence of variability between mother plants and regenerated plants from both the genotypes. Since these plantlets recovered from tolerant calli/embryoids/cell clumps selected from the medium supplemented with PEG6000, the possibility exists that these plants may prove to be tolerant against drought stress.

Formononetin Production by Large-Scale Cell Suspension Cultures of Medicago sativa L.

In this study, calli of Medicago sativa L. cv. Elçi (alfalfa Elçi) were inoculated in cell suspension culture and analyzed for aggregate assay, cell viability test, total phenolic content assay, DPPH free radical scavenging activity and formononetin assay by means of High-Performance Liquid Chromatography (HPLC). Hypocotyl, cotyledon and apical meristem explants were taken from 15-day-old aseptic seedlings and germinated in MS medium. 10 g calli were grown for each explant and then transferred into cell suspension culture. The highest cell viability rate, which was 75%, and the highest DPPH free radical scavenging activity with 51.36% was measured in 1000 mL cell suspension culture, while the highest total phenolic content, i.e. 40.2 mg/g, was quantified in 250 mL cell suspension culture. In accordance with the findings of the study, the production of formononetin was higher in the calli derived from cell suspension cultures than in herb samples of M. sativa. Moreover, in 1000 mL cell suspension culture, 4.99 mg/g of formononetin concentration was quantified, which scored the highest. In large-scale cell suspension cultures of M. sativa, it was possible to increase the production of formononetin production. Hence, due to its medicinal significance, a method has been tested to obtain higher amounts of this compound.

In vitro regeneration of Sesbania grandiflora using explants

Sesbania grandiflora is a fast-growing multipurpose legume tree used for animal feed, besides that. S. grandiflora has a high crude protein content. Several studies have reported regeneration of plantlets from explants but a successful transfer of regenerated plantlets following micropropagation of S. grandiflora has not previously been reported. The objective of this study was to regenerate S. grandiflora by tissue culture. Explants were cultured on Murashige Skoog (MS), Woody Plant Medium (WPM), and Gamborg medium (B5). The best medium was supplemented with different concentrations of naphthalene acetic acid (0.0, 0.08, 0.10 mg/l NAA) and butyric amino purine (1 mg/l BAP) for induction of shoots. Several concentrations of NAA (0.2, 0.4, 0,6 mg/l) were used to induce roots, and plantlets were then transplanted to acclimatization media. The basal medium, MS performed better than WPM and B5. The best shoot induction was obtained on MS medium containing 1 mg/l BAP and 0.08 mg/l NAA. Shootlets were rooted in WPM medium supplemented with 0.2 mg/l naphthalene acetic acid transplanted to acclimatization medium. MS medium and cotyledon explants can be used in micropropagation of turi.

An increased proportion of transgenic plants in the progeny of rapeseed (Brassica napus L.) transformants.

Cotyledon and leaf explants of two spring rapeseed varieties were transformed with Agrobacterium tumefaciens harboring a genetic construct with the gfp marker gene. In order to reduce the proportion of hyperhydrated shoots, which appeared during regenerant formation, we optimized sucrose content in the regeneration media. Analysis of the progeny obtained from T0 regenerants showed that in a number of lines the distribution of the gfp marker did not follow Mendelian segregation of a monogenic trait in self-pollinated plants, while in the progeny of the other lines of transgenic plants, the gfp marker was completely absent, although its presence had been confirmed in all selected T0 plants. We also found that in individual transformants gfp is randomly inherited throughout the central peduncle; its presence in the genome of seedlings does not depend on the location of the pod. Thus, both transformed and non-transformed cells were involved in the formation of gametes in T0 plants. In addition, marker segregation was different in plants of the T1 line obtained by nodal cuttings of a primary transformant, depending on the location of the cuttings on the stem of the original plant, indicating that the nature of T1 generation plants was also chimeric. Furthermore, we showed that propagation of plants by cutting followed by propagation by seeds formed as a result of self-pollination led to an increase in the proportion of transgenic plants in subsequent generations. The results obtained during the course of this study show that the transformants were chimeric, i. e. their tissues contained both transgenic and non-transgenic cells, and this chimeric nature was passed on to subsequent generations. We found that, in addition to nutrient media composition, other factors such as plant genotype and explant type also contribute to the rising of chimeric plants during transformation. Based on these results, we developed a simplified method, which consists of several rounds of a combination of cutting, seed production by self-pollination, and subsequent culling of wild-type plants, which significantly enriched descendent populations of the original rapeseed transformants with plants transgenic for the gfp marker

Effect of different growth regulators on in vitro regeneration of cotyledon explants of rough lemon (Citrus jambhiri Lush.) via callus induction

In order to establish in vitro protocols for raising aseptic cultures of cotyledon segments excised from five-week-old in vitro raised seedlings were used as explant material. Among surface sterilants, mercuric chloride 0.1% for three minutes provided least rate of contamination (24%) and the maximum explant survival (76%). The highest per cent culture establishment of 97.79% was obtained in cotyledon explants on Murashige and Skoog (MS) containing 2,4-D (3.0 mg/l) and maximum callus induction (95.41%) was observed on MS medium supplemented with 2,4-D (2.0 mg/l). Among different plant growth regulator treatments, maximum callus regeneration of 82.19% was observed on MS medium supplemented with BAP (3.0 mg/l) whereas, minimum callus regeneration (4.33%) was found in MS medium supplemented with BAP (3.0 mg/l) in combination with IAA (0.75 mg/l) and malt extract (500 mg/l). Maximum rooting response (96.00%) was recorded on MS (half strength) medium with NAA (2.0 mg/l) and was at par with rooting percentage of 92.00% on MS (half strength) medium supplemented with NAA (1.5 mg/l). The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (91.35%) was achieved in potting mixture containing garden soil with sand and farmyard manure (1:1:1).

Micropropagation through indirect organogenesis in Solanum capsicoides All., and assessment of clonal fidelity using ISSR markers

Solanum capsicoides All., is an important medicinal plant which is used as a source of ‘Kantakari’ (medicine for phlegm disorders) in India. The restricted distribution of the plant in Kerala along with its overexploitation necessitates the development of a protocol for in vitro propagation of S. capsicoides. The present study reports the effects of four different cytokinins (2 isopentenyl adenine (2iP), benzyl adenine (BAP), kinetin (KIN), and zeatin (ZEA)) in combination with indole 3-butyric acid (IBA) on indirect organogenesis of S. capsicoides from in vitro grown seedling explants such as cotyledon, shoot tip, and hypocotyl. Maximum shoot development was observed from cotyledon explants inoculated on MS medium supplemented with 2.46 μM IBA and 4.56 μM ZEA. An average of 17 ± 0.5 shoots per cotyledon with an average length of 0.53 ± 0.03 cm was observed. After subculture, an average of 20 ± 1 shoots was observed in cotyledon explants inoculated on Murashige and Skoog medium supplemented with 2.46 μM IBA and 6.84 μM ZEA. Clonal fidelity of regenerated plants was confirmed using DNA based molecular marker, intersimple sequence repeat (ISSR). Clonal fidelity test revealed 0% polymorphism and the PIC value 0 which indicate no diversity from seed-borne plants. The protocol thus developed can be used for rapid multiplication and conservation of S. capsicoides.

Requirements for efficient plantlet regeneration using cotyledonary nodal explants of purple coral tree (Erythrina fusca Lour.)

There are over hundred species in the genus Erythrina which are of practical importance in multiple ways. The requirements for Erythrina plantlet regeneration in vitro were rarely studied. Surface-sterilized seeds of purple coral tree (Erythrina fusca Lour.) exhibited a high germination rate (80%) in vitro. The cotyledonary nodes from the one-month-old purple coral tree seedlings grown in vitro were used as explants. About 70% of the cotyledonary nodal explants exhibited shoot regeneration when the explants were cultured vertically with the cut ends inserted into basal Murashige and Skoog (MS) medium compared to only 10% of those placed lying horizontally on the surface of the medium. Supplementation of the basal MS medium with 1 mg L−1 of BAP, 1 mg L−1 kinetin and 1 mg L−1 TDZ resulted in the greatest shoot elongation and the highest number of shoots developed from the cotyledonary nodal explants. The regenerated shoots were able to root (100%) on basal MS medium while supplementation with 2 mg L−1 of either NAA or IBA was inhibitory to root elongation. This is the first study on plantlet regeneration of purple coral tree which seems to have more complex requirements for shoot regeneration in vitro than those reported for the other three Erythrina species. Since only 30% of the rooted in vitro shoots survived during acclimatization following a previously published protocol for acclimatization of another plant, it is recommended that future studies are to investigate the requirements of higher plantlet survival for the rooted in vitro shoots of purple coral tree.

Enhancement of In Vitro Production of Volatile Organic Compounds by Shoot Differentiation in Artemisia spicigera.

Callus initiation, shoot formation and plant regeneration were established for Artemisia spicigera, a traditional medicinal plant growing in Armenia, Middle-Anatolia and Iran, and producing valuable volatile organic compounds (VOCs) that are mostly represented by monoterpenoids. Optimal callus initiation and shoot production were obtained by culture of hypocotyl and cotyledon explants on MS medium comprising 0.5 mg L−1 naphthalene acetic acid (NAA) and 0.5 mg L−1 6-benzyladenine (BA). Consequently, the shoots were transferred onto the MS media supplemented with 1 mg L−1 of indole-3-butyric acid (IBA) or 1 mg L−1 of NAA. Both types of auxin induced root formation on the shoots and the resulting plantlets were successfully grown in pots. The production of VOCs in callus tissues and regenerated plantlets was studied by gas chromatography–mass spectrometry (GC-MS) analysis. Although the potential of undifferentiated callus to produce VOCs was very low, an increased content of bioactive volatile components was observed at the beginning of shoot primordia differentiation. Intriguingly, the volatiles obtained from in vitro plantlets showed quantitative and qualitative variation depending on the type of auxins used for the rooting process. The acquired quantities based on total ion current (TIC) showed that the regenerated plantlets using 1 mg L−1 NAA produced higher amounts of oxygenated monoterpenes such as camphor (30.29%), cis-thujone (7.07%), and 1,8-cineole (6.71%) and sesquiterpene derivatives, namely germacrene D (8.75%), bicyclogermacrene (4.0%) and spathulenol (1.49%) compared with the intact plant. According to these findings, in vitro generation of volatile organic compounds in A. spicigera depends on the developmental stages of tissues and may enhance with the formation of shoot primordia and regeneration of plantlets.

Iron and zinc oxide nanoparticles: An efficient elicitor to enhance trigonelline alkaloid production in hairy roots of fenugreek

Fenugreek (Trigonella foenum-graecum L.) is a rich source of important medicinal metabolites. This plant belongs to the Fabaceae family that grows anywhere around the world. It is extensively used in medicine and as a food additive. Several secondary metabolites are detected in fenugreek, especially a valuable alkaloid called trigonelline. Enhancing the production of secondary metabolites is possible through the application of modern biotechnological techniques such as culturing hairy roots. In the present study, different strains of Agrobacterium rhizogenes (ATCC11325, ATCC15834, A4, A7, A13, and K599) were used for hairy root induction from fenugreek cotyledon explants. Then, the effect of various levels (x, 2x, and 4x concentrations of B5 medium) of iron oxide and zinc oxide nanoparticles were evaluated on growth, total phenolic, flavonoids, and trigonelline content of hairy roots. The growth curve analysis revealed an increase in the fresh and dry weight of treated hairy roots, as compared with the control. The highest levels of fresh (1.49 g) and dry weight (1.23 g) were obtained in hairy roots which were induced by the ATCC11325 strain and were treated with Zn (2x). The results indicated that using Zn nanoparticles on hairy roots can lead to higher levels of trigonelline, compared with the control group (i.e. transformed hairy roots without NPs).

Transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein imparts resistance to Maruca vitrata legume pod borer.

Fertile independent transgenic cowpea lines expressing the BtCry2Aa toxin with increased resistance to the most devastating lepidopteran insect pest, Maruca pod borer has been developed for the first time. Cowpea is a staple legume important for food and nutritional security in sub-Saharan Africa and Asia, where its production is limited by the key pest, legume pod borer (Maruca vitrata). Cowpea varieties resistant to M. vitrata are not known, hence, development of Maruca pod borer resistance cowpea through genetic engineering is a promising approach to improve its production. In the present study, transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein were developed for the first time using Agrobacterium tumefaciens-mediated transformation of cotyledonary explants. T0 plants recovered from Agrobacterium cocultured explants on medium containing 120 mgl-1 of kanamycin were identified on the basis of the presence of transgenes by PCR, their integration into genome by Southern hybridization and expression of their transcripts by semi quantitative PCR (sqRT-PCR) and quantitative Real-time-PCR (qRT-PCR) and protein by Western blot analysis. The transformation efficiency obtained was 3.47% with 11 independent T0 transgenic lines. The bioefficacy of Cry2Aa protein expressed in randomly selected four T0 plant's leaves and pods was evaluated by feeding Maruca pod borer demonstrated a significant lower damage and a high level of Maruca mortality (more than 90%) for all these Bt lines. The inheritance of transgenes from T0 to T1 progeny plants was demonstrated by PCR analysis. The transgenic plants generated in this study can be used in cowpea breeding program for durable and sustainable legume pod borer resistance.

In vitro morphogenesis of Physalis ixocarpa Brot ex. Horm

ABSTRACT Physalis ixocarpa Brot. ex Horm. is considered the most economically important species of the genus. Tissue culture is pointed out as a strategy for its propagation, but researches indicate that in vitro responses are genotype-dependent. This study aimed to evaluate the in vitro morphogenesis of the P. ixocarpa green and purple varieties, in view of the massive propagation of the species. The morphogenic capacity of the explants cotyledonary node, hypocotyl and cotyledon was evaluated in Murashige & Skoog medium supplemented with benzylaminopurine - BAP (0.00, 2.5, 5.0, 7.5 or 10.0 μM) and naphthaleneacetic acid - NAA (0.00 or 2.5 μM), using a completely randomized experimental design, in a 3 x 5 x 2 factorial scheme, with 30 treatments for each variety. The number of shoots per direct and indirect organogenesis and the percentage of explants with callus were analyzed. The in vitro morphogenetic expression of P. ixocarpa is influenced by the type of explant and by the plant regulators BAP and NAA. The cotyledonary node explant is efficient for the production of shoots via direct organogenesis in the two varieties studied.